Blood cells have a relatively short life span and need to be replenished throughout life. In adults, blood cell formation or hematopoiesis takes place in the bone marrow, but blood-forming stem cells can also be found in peripheral blood. Hematopoietic cells represent a hierarchy of proliferating and differentiating cells. The most abundant are the differentiating or lineage committed cells. These cells have limited or no proliferative capacity and represent specialized end cells that are found in blood, and their immediate precursors.
The immediate precursors of the differentiating cells are the progenitor cells. Most of these cells are restricted to differentiate along a single lineage but they may have quite extensive proliferative capacity. Progenitor cells appear morphologically as blast cells, and they typically do not have specific features of the hematopoietic lineage to which they are committed.
Progenitor cells are derived from stem cells. Stem cells have been historically defined as cells capable of long term hematopoietic repopulation. This implies their ability to self-renew as well as to generate daughter cells of any of the hematopoietic lineages. The presence of stem and progenitor cells may be detected by their ability to produce colony-forming cells in culture and repopulate xenogeneic hosts such fetal sheep (Zanjani et al., 1994 J. Clin. Invest, Vol. 89, p. 1178-1188) and immuno-deficient mice (Dick et al., 1991 Immunological Reviews, Vol. 124:25-43). They may also be detected by screening for the CD34 antigen which is a positive marker for early hematopoietic cells including colony forming cells and stem cells. At present, the long term culture initiating cell (LTCIC) assay appears to be the best way to detect stem cells, or at least the most primitive progenitor cells, using tissue culture methodologies.
There is a continued interest in developing stem cell purification techniques. Pure populations of stem cells will facilitate studies of hematopoiesis. Transplantation of hematopoietic cells from peripheral blood and/or bone marrow is also increasingly used in combination with high-dose chemo- and/or radiotherapy for the treatment of a variety of disorders including malignant, nonmalignant and genetic disorders. Very few cells in such transplants are capable of long-term hematopoietic reconstitution, and thus there is a strong stimulus to develop techniques for purification of hematopoietic stem cells. Furthermore, serious complications and indeed the success of a transplant procedure is to a large degree dependent on the effectiveness of the procedures that are used for the removal of cells in the transplant that pose a risk to the transplant recipient. Such cells include T lymphocytes that are responsible for graft versus host disease (GVHD) in allogenic grafts, and tumor cells in autologous transplants that may cause recurrence of the malignant growth. It is also important to debulk the graft by removing unnecessary cells and thus reducing the volume of cyropreservant to be infused.
Hematopoietic cells have been separated on the basis of physical characteristics such as density and on the basis of susceptibility to certain pharmacological agents which kill cycling cells. The advent of monoclonal antibodies against cell surface antigens has greatly expanded the potential to distinguish and separate distinct cell types. There are two basic approaches to separating cell populations from bone marrow and peripheral blood using monoclonal antibodies. They differ in whether it is the desired or undesired cells which are distinguished/labeled with the antibody(s).
In positive selection techniques the desired cells are labeled with antibodies and removed from the remaining unlabeled/unwanted cells. In negative selection, the unwanted cells are labeled and removed. Antibody/complement treatment and the use of immunotoxins are negative selection techniques, but FACS sorting and most batch wise immunoadsorption techniques can be adapted to both positive and negative selection. In immunoadsorption techniques cells are selected with monoclonal antibodies and preferentially bound to a surface which can be removed from the remainder of the cells e.g. column of beads, flasks, magnetic particles. Immunoadsorption techniques have won favour clinically and in research because they maintain the high specificity of targeting cells with monoclonal antibodies, but unlike FACSorting, they can be scaled up to deal directly with the large numbers of cells in a clinical harvest and they avoid the dangers of using cytotoxic reagents such as immunotoxins, and complement.
Current positive selection techniques for the purification of hematopoietic stem cells target and isolate cells which express CD34 (approximately 1-2% of normal bone marrow) (Civin, C. l., Trischmann, T. M., Fackler, M. J., Bernstein, I. D., Buhring, H. J., Campos, L. et al. 1989 Report on the CD34 cluster workshop, In: Leucocyte typing IV, White Cell Differentiation Antigens. Knapp, W., Dorken, B., Gilks, W. R., Reiber, E P., Schmidt, R. E., Stein, H., and Kr. von den Borne, A. E. G Eds., Oxford University Press. Oxford, pp.818). Thus, the potential enrichment of hematopoietic stem cells using this marker alone is approximately 50 fold. Available techniques typically recover 30-70% of the CD34.sup.+ cells in the start suspension and produce an enriched suspension which is 50-90% CD34.sup.+ (Firat et al., 1988, Bone Marrow Transplantation, Vol. 21:933-938; deWynter, E. A. et al., 1975, Stem Cells, Vol. 13:524-532; Shpall, E. J., et al. 1994, J. of Clinical Oncology 12:28-36; Thomas, T. E., 1994, Cancer Research, Therapy and Control 4(2): 119-128). The positive selection procedures suffer from many disadvantages including the presence of materials such as antibodies and/or magnetic beads on the CD34.sup.+ cells, and damage to the cells resulting from the removal of these materials. Also to be considered is the recent evidence that some long term repopulating cells are CD34.sup.- (negative) (Zanjani et al., 1998, Exp. Hematol., Vol. 26:353-360) and methods that isolate CD34.sup.+ will not capture these cells.
Negative selection has been used to remove minor populations of cells from clinical grafts. These cells are either T-cells or tumor cells that pose a risk to the transplant recipient. The efficiency of these purges varies with the technique and depends on the type and number of antibodies used. Typically, the end product is very similar to the start suspension, missing only the tumor cells or T-cells.
Transplants of purified stem cells without differentiated or lineage committed cells will give short and long-term hematopoietic support (Shpall, E. J., et al. 1994, J. of Clinical Oncology 12:28-36). Since differentiated cells make up a vast majority of the cells in bone marrow and blood, depletion of these cells produces a much smaller cell suspension. The number of cells in the final product and the degree of enrichment of progenitor/stem cells will depend on the efficiency of the antibody targeting and the removal of labeled cells.
There are several studies that enrich for hematopoietic stem cells by depleting lineage committed cells but all require a number of positive or negative selection steps to achieve the desired degree of enrichment (50 fold). Early studies required prior density separation and extensive incubations to remove adherent cells (Linch, D. C, and Nathan, D. G. 1984, Nature 312 20/27: 775-777; Sieff, C. A., et al., 1985, Science 230: 1171-1173; Kannourakis, G. and Bol, S., 1987 Exp. Hematol 15:1103-1108.). More recent techniques are no less cumbersome; involving density separation steps followed by two partial lineage depletions (Winslow, J. M., et al., 1994, Bone Marrow Transplantation 14:265-271) or a partial lineage depletion using panning or FACS followed finally by positive selection using FACS (Carlo-Stella et al. 1994, Blood 84, 10 supple.:104a; Reading, C., et al. (1994), Blood 84, 10 supple.:399a). Most of these methods for lineage depletion lack effective antibody combinations against myeloid cells, erythrocytes and/or B-cells.
U.S. Pat. No. 5,087,570 describes a process for preparing a hematopoietic cell composition using a combination of positive and negative selection. The process relies on the use of antibody to the Sca-1 antigen which is associated with murine clonogenic bone marrow precursors of thymocytes and progeny T-cells. The Sca-1 antibody is not useful in isolating human hematopoietic cells.
Epithelial cancers of the bronchi, mammary ducts and the gastrointestinal and urogenital tracts represent a major type of solid tumors seen today. Micrometastatic tumor cell migration is thought to be an important prognostic factor for patients with epithelial cancer (Vaughan et al., 1990, Proc. Am. Soc. Clin. Oncol. 9:9). Our ability to detect such metastatic cells is limited by the effectiveness of tissue or fluid sampling and the sensitivity of tumor detection methods. From a research point of view, it is also very difficult to study such rare cells and determine the biological changes which enable spread of disease. Metastatic epithelial tumor cells disseminate to distant sites such as bone marrow and lymph nodes. Bone marrow has become an important indicator organ for the spread of epithelial cells because of its easy accessibility and the lack of normal epithelial cells making identification of tumor cells less difficult. The recent trend in autologous transplantation away from the use of bone marrow grafts to cytokine mobilized peripheral blood has raised the question of how often peripheral blood is contaminated with micrometastatic tumor cells. Epithelial tumor contamination in peripheral blood is less frequent than in bone marrow (Ross et al., 1993, Blood, 82(9):2605-2610) but cytokine mobilization may also "mobilize" tumor cells (Brugger et al., 1994, Blood, 83(3):636-640). Both cancer research and patient therapy could benefit from method of enriching epithelial tumor cells from blood, bone marrow and peritoneal and pleural effusions.
The two most poplar methods in research laboratories for the detection of rare epithelial tumor cells are immuno-cytochemical staining (ICC) and polymerase chain reaction (PCR). PCR detects specific DNA or RNA sequences. ICC methods rely on antibodies to epithelial-specific cytoskeleton and membrane antigens to stain tumor cells. ICC is more widely used clinically and established laboratories with experienced staff are consistently reporting sensitivities of one tumor cell is 10.sup.5 bone marrow cells (Pantel, 1996, J. of Hematotherapy, 5:359-367). An enrichment of 100 fold or 2 log could increase this sensitivity to one in 10.sup.7 cells.
There are two approaches to enriching epithelial tumor cells from a suspension of non-epithelial cells such as bone marrow or blood. One can either target the tumor cells for recovery using an epithelial or tumor specific antibodies (positive selection) or target all the non-epithelial (in this case hematopoietic cells) for depletion (negative selection). The problems with the fist approach, positive selection, is that the recovered tumor cells are covered with antibodies and the sites commonly used for immunocytochemical detection are blocked. It is also difficult to positively select cells from samples that have been stored or previously frozen. The non-specific binding of antibodies to cells or of cells to the separation matrix are too high. Negative selection, on the other hand, can deal with clumpy or previously frozen cell suspensions (Thomas et al., 1998, Methods in Enzymology: Signalling Pathways and Gene Regulation in Hematopoietic Cell Growth and Differentiation "Purification of Hematopoietic Stem cells for Further Biological Study", Academic Press) as the recovered cells have not been labelled with antibody. Both currently available epithelial tumor cells enrichment methods are positive selections using cytokeratin specific antibodies or antibodies to Human Epithelial Antigen (HEA) (Miltenyi Biotec Inc. Aubum CA; and Dynal, Skoyen Norway). A negative selection technique that employs antibodies to CD45 has also been reported but enrichments are only 1-2 log and vary with cell source. Van Vlasselaer (U.S. Pat. No. 5,648,223) teaches a procedure for enriching tumor cells in whole blood using cell-trap centrifugation to enrich tumor cells in circulating bodily fluids, by separation based on density. However, the methods taught by Van Vlasselaer require the construction and operation of a cell trap centrifuge tube calibrated to specific gradients of density, osmolality and pH.
In order to successfully utilize circulating bodily fluids for cancer diagnosis, improved methods of enriching the small number of circulating tumor cells are required.